We have established human cell lines which elaborate mediators of in vitro hematopoiesis. Colony-stimulating activity (CSA) stimulates granulocyte-macrophage (CFU-GM) growth in humans and other species, promotes the growth of certain myelogenous leukemia cell populations and is similar to another mediator called erythropoiesis-enhancing activity (EEA). CSA, purified 1,000-fold from serum-free cell line conditioned medium, is sensitive to trypsin and to reduction and alkylation, suggesting a protein containing critical disulfide bonds. Gel filtration yields molecular species of 140,000 and 30,000 daltons which stimulate mouse marrow and are inactivated by an anti-human urinary colony-stimulating factor (CSF) antibody and a 30,000 dalton CSF for human marrow which is resistant to inactivation. Hydrophobic chromatography of the 30,000 dalton CSA results in virtually complete separation of a CSF promoting chiefly neutrophilic colony growth in human marrow from the remainder of CSA and EEA. We propose to purify the hydrophobic CSF and, if possible, other CSFs to homogeneity, utilizing such techniques as high-pressure liquid chromatography, antibody affinity and chromatofocusing in addition to older methods; radiolabel CSF(s); prepare anti-CSF polyclonal and monoclonal antibodies; and develop radioimmunoassays for the molecules. We will use these probes to study the effects of CSFs and other factors in cell line CM on the proliferation and differentiation of acute myelogenous leukemia cells in culture, comparing their effects with those of factors elaborated by the leukemia cells. By so doing, we will further characterize the molecular nature of CSFs and their role in normal and leukemic hematopoiesis and provide probes which may further delineate the role of CSFs in vitro.